1. Field of the Invention
This invention relates to immunoassay for a ligand, and more particularly relates to membrane immunoassay and particular reagents useful therein.
2. Background of the Invention
Assay systems which are both rapid and sensitive have been developed to determine the concentration of a substance, generally referred to as the analyte, present in low concentration in a fluid sample. Immunoassays depend on the binding of an antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. These assays employ one of the above reagents in labeled form, the labeled reagent being referred to as the tracer.
Enzymes have often been used as labels in immunoassay. In conventional enzyme immunoassay (EIA), an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a signal which is detected and measured. The signal may be a color change, detected with the naked eye or by a spectrophotometric technique, or may be conversion of the substrate to a product detected by fluorescence.
A convenient format for EIA is solid phase immunoassay in which one of the assay reagents is immobilized on a solid support. The solid support may be in the form of a dipstick, the inside wall of a test tube or cuvette or the well of a microtiter plate. A particularly useful solid support is a microporous membrane.
Membrane immunoassay is often referred to as flow-through assay. Examples of flow-through EIA wherein flow is generated by capillary action are the assays described in U.S. Pat. No. 3,888,629 to Bagshaw, U.S. Pat. No. 4,246,339 to Cole et al. and U.S. Pat. No. 4,632,901 to Valkirs et al. U.S. Pat. No. 4,277,560 to Gray and U.S. Pat. No. 4,812,293 to McLaurin et al. are examples of flow-through assays using pressure and vacuum respectively.
In membrane EIA, any number of liquids may be caused to flow through the membrane to effect binding, separation and washing of assay components. The final step in most membrane EIA procedures is contacting a color developing reagent, such as a chromogen, with the membrane. The chromogen reacts with enzyme captured on the membrane to produce a colored product which may be detected as evidence of the presence of analyte or measured as evidence of the concentration of analyte. The colored product may be soluble, in which case it will pass through the membrane and be detected in the filtrate, or it may be insoluble and form a colored spot on the membrane.
The enzyme urease converts urea into carbon dioxide and ammonia. It has been developed as a label for solution immunoassay wherein a rise in pH of the assay medium due to the ammonia production is detected colorimetrically with an indicator such as bromcresol purple (Chandler et al., Journal of Immunological Methods 53,187 (1982); U.S. Pat. No. 4,590,157).
EIA in which urease is detected colorimetrically in solution by the Chandler et al. procedure provides an excellent visual readout because the detected product is deeply colored and water soluble. On the other hand, rapid diffusion due to the water solubility precludes deposition of the product as a spot on a solid phase, such as a dipstick or a membrane. This severely limits usefulness of urease in solid phase EIA procedures. There is a need for a urease substrate which would be converted to an insoluble product to be deposited on a solid phase. Such a substrate would greatly extend the usefulness of urease as an immunological label. The present invention provides such a substrate.